JP4245746B2 - Amino acid production by fermentation - Google Patents
Amino acid production by fermentation Download PDFInfo
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- JP4245746B2 JP4245746B2 JP26510899A JP26510899A JP4245746B2 JP 4245746 B2 JP4245746 B2 JP 4245746B2 JP 26510899 A JP26510899 A JP 26510899A JP 26510899 A JP26510899 A JP 26510899A JP 4245746 B2 JP4245746 B2 JP 4245746B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Abstract
Description
【0001】
【発明の属する技術分野】
本願発明は、発酵法によるアミノ酸の工業的に効率の良い製造法に関する。
【0002】
【従来の技術】
糖から直接L−アミノ酸を生成蓄積させる直接発酵法としては、コリネバクテリウム属、ブレビバクテリウム属、エシェリヒア属、セラチア属、アースロバクター属などの微生物の野生株から誘導した突然変異株を用いる方法が知られている。例えばL−アミノ酸生産性変異株としては、アミノ酸等の栄養要求性を有した菌株(特公昭56-10037)、アミノ酸のアナログやビタミン等の耐性変異を有した菌株(特開昭56-134993、特開昭62-44193)、栄養要求性変異とアミノ酸のアナログ耐性変異を共有した菌株(特開昭50-31093、特開昭56-134993)、分解能の低下した菌株(特開昭63-273487、特公昭52-48195)、あるいはアミノアシルt−RNA合成酵素に基質親和性が減少した変異を有する菌株(特開平4-330275)等が知られている。
【0003】
さらにアミノ酸の生合成に係わる遺伝子を含む組換え体DNAで形質転換された菌株を用いることで、アミノ酸の生産性を向上することができることが知られている(特開昭58-893、特開昭60-12995、特開昭60-210994、特開昭60-30693、特開昭61-195695、特開昭61−271981、特開平2-458、特開平2-42988、特公平1-42676、特公平5-11960、特公平5-26467)。
【0004】
L−トリプトファンの製造においては、アミノキノリン誘導体またはフェノチアジン誘導体に対する耐性を付与することでアミノ酸生産性を向上させた例が報告されている(特開平4−112795)。
【0005】
【発明が解決しようとする課題】
本願発明の目的は、医薬品、化学品、食品および飼料添加物などとして有用なアミノ酸の工業的に効率のよい製造方法を提供することにある。
【0006】
【課題を解決するための手段】
即ち、本願発明は、以下の(1)〜(10)に関する。
(1) L−アラニン、L−バリン、L−ロイシン、L−イソロイシン、L−メチオニン、L−フェニルアラニン、L−プロリン、グリシン、L−セリン、L−トレオニン、L−システイン、L−チロシン、L−アスパラギン、L−グルタミン、L−リジン、L−ヒスチジン、L−アルギニン、L−アスパラギン酸およびL−グルタミン酸からなる群より選ばれるアミノ酸(以下、本願発明にかかわるアミノ酸と略す)の生産能を有し、かつアミノキノリン誘導体に対する耐性を有する微生物を培地に培養し、培養物中に該アミノ酸を生成蓄積させ、該培養物から該アミノ酸を採取することを特徴とする、該アミノ酸の製造法。
(2) アミノキノリン誘導体が、クロロキン、アモジアキン、ペンタキン、プリマキンおよびこれら物質のアルカリ金属塩からなる群より選ばれるアミノキノリン誘導体である、上記(1)のアミノ酸の製造法。
(3) アミノ酸がL−ヒスチジンである、上記(1)のアミノ酸の製造法。
【0007】
(4) 微生物がセラチア属、コリネバクテリウム属、アースロバクター属、ミクロバクテリウム属、バチルス属、エシェリヒア属からなる群より選ばれる微生物である、上記(1)のアミノ酸の製造方法。
(5) 微生物がエシェリヒア・コリH−9341(FERM BP−6674)である、上記(4)のアミノ酸の製造方法。
(6) 本願発明にかかわるアミノ酸の生産能を有し、かつアミノキノリン誘導体に対する耐性を有する微生物。
(7) アミノキノリン誘導体が、クロロキン、アモジアキン、ペンタキン、プリマキンおよびこれら物質のアルカリ金属塩からなる群より選ばれるアミノキノリン誘導体である、上記(6)の微生物。
(8) アミノ酸がL−ヒスチジンである、上記(6)の微生物。
(9) 微生物がセラチア属、コリネバクテリウム属、アースロバクター属、ミクロバクテリウム属、バチルス属、エシェリヒア属からなる群より選ばれる微生物である、上記(6)〜(8)いずれか1つに記載の微生物。
(10) エシェリヒア・コリH−9341(FERM BP−6674)。
【0008】
【発明の実施の形態】
本願発明にかかわるアミノ酸の生産菌(以下、本願発明の微生物という)としては、本願発明にかかわるアミノ酸の生産能を有し、かつアミノキノリン誘導体に対する耐性を有する微生物であればいずれも用いることができる。該微生物としては、例えば、セラチア属、コリネバクテリウム属、アースロバクター属、ミクロバクテリウム属、バチルス属、エシェリヒア属に属する微生物、例えばSerratia ficaria、Serratia fonticola、Serratia liquefaciens、Serratia marcescens、Corynebacterium glutamicum、Corynebacterium mycetoides、Corynebacterium variabilis、Corynebacterium ammoniagenes、Arthrobacter crystallopoietes、Arthrobacter duodecadis、Arthrobacter ramosus、Arthrobacter sulfureus、Arthrobacter aurescens、Arthrobacter citreus、Arthrobacter globiformis、Microbacterium ammoniaphilum、Bacillus subtilis、Bacillus amyloliquefacines、Escherichia coli等をあげることができる。
【0009】
本願発明に用いられるアミノキノリン誘導体は、アミノキノリン骨格を有する物質であればいずれでも用いられる。例えば、クロロキン(chloroquine)、アモジアキン(amodiaquine)などの4-アミノキノリン誘導体や、ペンタキン(pentaquine)、プリマキン(primaquine)などの8-アミノキノリン誘導体などが用いられる。また、これら物質のアルカリ金属塩などの塩も用いられる。これら物質は、いずれも抗マラリア薬(antimalarial drugs)として知られている。ここで、アルカリ金属はナトリウム、カリウム等のアルカリ金属であればいずれでも用いられる。
【0010】
本願発明の微生物は、本願発明にかかわるアミノ酸を生産する能力を有する微生物に紫外線照射やN−メチル−N′−ニトロ−N−ニトロソグアニジン(NTG)などの突然変異誘発剤による通常の変異処理を施し、該変異株を親株が生育できないか、または生育が不良となる濃度のアミノキノリン誘導体を含む寒天平板培地上で通常の条件下で培養し、生じたコロニーのうちで親株よりも生育が速いか大きなコロニーを生ずる株を選択することにより、アミノキノリン誘導体に対する耐性の付与された株として取得することができる。
【0011】
上記のアミノ酸生産能を有する微生物として、本願発明にかかわるアミノ酸の生産能を有する微生物を用いても良いし、また、公知の方法により新たに野生型株から取得しても良い。公知の方法としては、上記の変異処理法の他に、細胞融合法、形質導入法、その他の遺伝子組換え技法[いずれもMolecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)(以下、モレキュラー・クローニング第2版と略す)]等をあげることができる。
【0012】
また、本願発明の微生物は、アミノキノリン誘導体に対する耐性を有する微生物を通常の変異処理法を用いて取得した後、該微生物に本願発明にかかわるアミノ酸生産能を上記方法により付与することによっても取得することができる。
本願発明の微生物として、具体的にはエシェリヒア・コリH−9341(FERM BP−6674)をあげることができる。
【0013】
本願発明の微生物を用いた本願発明にかかわるアミノ酸の生産は、通常の細菌培養法にて実施することができる。
本願発明にかかわるアミノ酸の生産に用いる培地としては、炭素源、窒素源、無機物、その他使用菌株の必要とする微量の栄養素を程よく含有するものならば、合成培地または天然培地いずれも使用可能である。
【0014】
炭素源としては、グルコース、フラクトース、ラクトース、糖蜜、セルロース加水分解物、粗糖加水分解物、澱粉加水分解物などの炭水化物、ピルビン酸、酢酸、フマル酸、リンゴ酸、乳酸などの有機酸、グリセリン、エタノールなどのアルコールなどが用いられる。
窒素源としては、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸アンモニウムなどの各種無機塩類、有機酸のアンモニウム塩、アミン類、ペプトン、肉エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕加水分解物、各種発酵菌体およびその消化物などが用いられる。
【0015】
無機物としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、塩化カルシウム、炭酸カルシウムなどが用いられる。
培養は、振盪培養または通気撹拌培養などの好気的条件下にて行われ、培養温度は20〜40℃で、好ましくは28〜37℃の範囲である。培地のpHはpH5〜9の範囲で、好ましくは中性付近に保持する。培地のpH調整は炭酸カルシウム、無機あるいは有機の酸、アルカリ溶液、アンモニア、pH緩衝液などによって行う。通常1〜7日間の培養により、培養液中に本願発明にかかわるアミノ酸が生成蓄積する。
【0016】
培養終了後、培養液から菌体などの沈殿物を除去し、イオン交換処理法、濃縮法、塩析法などを併用することにより、培養液から該アミノ酸を回収することができる。
本願発明により得られるアミノ酸としては先にあげた本願発明にかかわるアミノ酸であれば特に限定されないが、例えばL−ヒスチジンをあげることができる。
以下に本願発明の実施例を示すが、本願発明はこれらの実施例に限定されるものではない。
【0017】
【実施例】
実施例1 アミノキノリン誘導体に対する耐性を有するL−ヒスチジン生産性変異株の取得
ブタペスト条約に基いて平成11年 3月9日付けで、FERM BP−6673 として工業技術院生命工学工業技術研究所(日本国茨城県つくば市東1丁目1番3号)に寄託されている、メチオニン要求性のエシェリヒア・コリATCC21318より誘導した1,2,4−トリアゾールアラニンに対する耐性を有するL−ヒスチジン生産性変異株H−9340に、常法に従って、N−メチル−N′−ニトロ−N−ニトロソグアニジン(NTG)による変異処理(0.2mg/ml、30℃、30分間)を施した後、プリマキン二ナトリウム塩を150mg/L含む寒天平板培地〔グルコース 0.2%、リン酸一カリウム 0.3%、リン酸二ナトリウム 0.6%,硫酸マグネシウム 0.01%、塩化ナトリウム 0.05%,塩化アンモニウム 0.1%、 栄養要求物質(DL−メチオニン)50mg/L、寒天 1.5%、pH7.2〕に塗布した。
【0018】
菌体を塗布した上記寒天平板培地を30℃で2〜6日間培養し、生育してきた大きなコロニーを釣菌分離し、H−9341を取得した。この菌株はブタペスト条約に基いて平成11年3月9日付けで、FERM BP−6674 として工業技術院生命工学工業技術研究所(日本国茨城県つくば市東1丁目1番3号)に寄託されている。
実施例2 プリマキンを含む寒天平板培地での生育比較試験
実施例1で取得した変異株H−9341のプリマキン含有寒天平板培地での生育を親株H−9340と比較した。
【0019】
それぞれの変異株を取得した時と同濃度のプリマキン二ナトリウム塩を含む上記寒天平板培地上に、天然培地で24時間培養した各菌体を生理食塩水に懸濁して1〜10cells/cm2になるように塗布し、33℃、4日間培養した。
該培養による生育の可否を第1表に示した。
親株であるH−9340は、プリマキンを含む寒天培地上では生育できなかった。
【0020】
【表1】
【0021】
実施例3 L−ヒスチジンの製造
実施例1で取得した変異株H−9341または親株H−9340を用いたL−ヒスチジンの製造を以下の方法で行った。
H−9340とH−9341を、それぞれ太型試験管中の種培地(グルコース2%、糖蜜0.5%、コーンスチープリカー 1%、硫安 1.2%、リン酸一カリウム 0.3%、硫酸マグネシウム 0.015%、DL−メチオニン 600mg/L、アデニン 100mg/L、炭酸カルシウム 3%、pH6.2)6mlに接種して、30℃で12時間振盪培養した。
【0022】
得られた種培養液0.1mlを、それぞれ太型試験管中の生産培地(グルコース 6%、コーンスチープリカー 1%、硫酸アンモニウム 2.4%、リン酸一カリウム 0.4%、硫酸マグネシウム 0.015%、チアミン塩酸塩 10mg/L、パントテン酸カルシウム 10mg/L、炭酸カルシウム 3%、pH6.5)5mlに接種して、30℃で48時間振盪培養した。
【0023】
培養後、培養液中のL−ヒスチジンの蓄積量を、高速液体クロマトグラフィー法により定量した。
結果を第2表に示した。
親株H−9340に比べ変異株H−9341のL−ヒスチジン生産性は向上していた。
【0024】
【表2】
【0025】
【発明の効果】
本願発明により、L−アラニン、L−バリン、L−ロイシン、L−イソロイシン、L−メチオニン、L−フェニルアラニン、L−プロリン、グリシン、L−セリン、L−トレオニン、L−システイン、L−チロシン、L−アスパラギン、L−グルタミン、L−リジン、L−ヒスチジン、L−アルギニン、L−アスパラギン酸およびL−グルタミン酸からなる群より選ばれるアミノ酸(以下、本願発明にかかわるアミノ酸と略す)の生産能を有し、かつアミノキノリン誘導体に対する耐性を有する微生物を取得し、培地で培養することで、本願発明にかかわるアミノ酸の生産性を向上させることができ、本願発明にかかわるアミノ酸を工業的に効率よく、かつ安価に製造することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an industrially efficient production method of amino acids by fermentation.
[0002]
[Prior art]
As a direct fermentation method for directly producing and accumulating L-amino acids from sugar, a mutant strain derived from a wild strain of a microorganism such as Corynebacterium, Brevibacterium, Escherichia, Serratia, or Arthrobacter is used. The method is known. For example, L-amino acid-producing mutant strains include strains having auxotrophy such as amino acids (Japanese Patent Publication No. 56-10037), strains having resistance mutations such as amino acid analogs and vitamins (Japanese Patent Laid-Open No. 56-134993, JP-A 62-44193), strains sharing auxotrophic mutations and amino acid analog resistance mutations (JP-A 50-31093, JP-A 56-134993), strains with reduced resolution (JP-A 63-273487) Japanese Patent Publication No. 52-48195), or a strain having a mutation with reduced substrate affinity in aminoacyl t-RNA synthetase (Japanese Patent Laid-Open No. 4-330275).
[0003]
Furthermore, it is known that amino acid productivity can be improved by using a strain transformed with a recombinant DNA containing a gene involved in amino acid biosynthesis (JP 58-893, JP JP-A-60-12995, JP-A-60-210994, JP-A-60-30693, JP-A-61-195695, JP-A-61-271981, JP-A-2-45888, JP-A-2-42988, JP-B-1-42676 , JP-5-11960, JP-B-5-26467).
[0004]
In the production of L-tryptophan, an example in which amino acid productivity is improved by imparting resistance to an aminoquinoline derivative or phenothiazine derivative has been reported (Japanese Patent Laid-Open No. 4-12795).
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide an industrially efficient method for producing amino acids useful as pharmaceuticals, chemicals, foods, feed additives and the like.
[0006]
[Means for Solving the Problems]
That is, this invention relates to the following (1)-(10).
(1) L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, glycine, L-serine, L-threonine, L-cysteine, L-tyrosine, L -Has the ability to produce an amino acid selected from the group consisting of asparagine, L-glutamine, L-lysine, L-histidine, L-arginine, L-aspartic acid and L-glutamic acid (hereinafter abbreviated as amino acids according to the present invention). And a method for producing the amino acid, comprising culturing a microorganism having resistance to an aminoquinoline derivative in a medium, producing and accumulating the amino acid in the culture, and collecting the amino acid from the culture.
(2) The method for producing an amino acid according to (1) above, wherein the aminoquinoline derivative is an aminoquinoline derivative selected from the group consisting of chloroquine, amodiaquine, pentaquine, primaquine and alkali metal salts of these substances.
(3) The method for producing an amino acid according to (1) above, wherein the amino acid is L-histidine.
[0007]
(4) The method for producing an amino acid according to (1) above, wherein the microorganism is a microorganism selected from the group consisting of Serratia, Corynebacterium, Arthrobacter, Microbacterium, Bacillus, and Escherichia.
(5) The method for producing an amino acid according to (4) above, wherein the microorganism is Escherichia coli H-9341 (FERM BP-6673).
(6) A microorganism having the ability to produce amino acids according to the present invention and having resistance to aminoquinoline derivatives.
(7) The microorganism according to (6) above, wherein the aminoquinoline derivative is an aminoquinoline derivative selected from the group consisting of chloroquine, amodiaquine, pentaquine, primaquine, and alkali metal salts of these substances.
(8) The microorganism according to (6) above, wherein the amino acid is L-histidine.
(9) Any one of the above (6) to (8), wherein the microorganism is a microorganism selected from the group consisting of Serratia, Corynebacterium, Arthrobacter, Microbacterium, Bacillus, and Escherichia The microorganism described in 1.
(10) Escherichia coli H-9341 (FERM BP-6673).
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Any amino acid-producing bacterium according to the present invention (hereinafter referred to as the microorganism of the present invention) can be used as long as it has the ability to produce amino acids according to the present invention and has resistance to aminoquinoline derivatives. . Examples of the microorganism include, for example, Serratia, Corynebacterium, Arthrobacter, Microbacterium , Bacillus, Escherichia, such as Serratia ficaria , Serratia fonticola , Serratia liquefaciens , Serratia marcescens , Corynebacterium glutamicum , Corynebacterium mycetoides, Corynebacterium variabilis, Corynebacterium ammoniagenes , Arthrobacter crystallopoietes, Arthrobacter duodecadis, Arthrobacter ramosus, Arthrobacter sulfureus, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globiformis, Microbacterium ammoniaphilum, Bacillus subtilis, Bacillus amyloliquefacines, can be mentioned Escherichia coli and the like.
[0009]
As the aminoquinoline derivative used in the present invention, any substance having an aminoquinoline skeleton may be used. For example, 4-aminoquinoline derivatives such as chloroquine and amodiaquine, and 8-aminoquinoline derivatives such as pentaquine and primaquine are used. Further, salts such as alkali metal salts of these substances are also used. All of these substances are known as antimalarial drugs. Here, any alkali metal may be used as long as it is an alkali metal such as sodium or potassium.
[0010]
The microorganism of the present invention is subjected to normal mutation treatment with a mutagen such as ultraviolet irradiation or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) on the microorganism having the ability to produce the amino acid according to the present invention. The mutant strain is cultured under normal conditions on an agar plate medium containing an aminoquinoline derivative at a concentration at which the parent strain cannot grow or grows poorly, and the resulting colonies grow faster than the parent strain. By selecting a strain producing such a large colony, it can be obtained as a strain imparted with resistance to aminoquinoline derivatives.
[0011]
As the above microorganism having amino acid-producing ability, a microorganism having amino acid-producing ability according to the present invention may be used, or newly obtained from a wild-type strain by a known method. Known methods include cell fusion, transduction, and other gene recombination techniques in addition to the above-described mutation treatment methods [all are Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (Hereinafter abbreviated as "Molecular Cloning 2nd Edition").
[0012]
In addition, the microorganism of the present invention can be obtained by obtaining a microorganism having resistance to an aminoquinoline derivative using a usual mutation treatment method and then imparting the microorganism with the ability to produce an amino acid according to the present invention by the above method. be able to.
A specific example of the microorganism of the present invention is Escherichia coli H-9341 (FERM BP-6664).
[0013]
The production of amino acids according to the present invention using the microorganism of the present invention can be carried out by a conventional bacterial culture method.
As a medium used for the production of amino acids according to the present invention, either a synthetic medium or a natural medium can be used as long as it contains moderate amounts of nutrients required by the carbon source, nitrogen source, inorganic substance, and other strains used. .
[0014]
As carbon sources, carbohydrates such as glucose, fructose, lactose, molasses, cellulose hydrolyzate, crude sugar hydrolyzate, starch hydrolyzate, organic acids such as pyruvic acid, acetic acid, fumaric acid, malic acid, lactic acid, glycerin, Alcohol such as ethanol is used.
Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic salts, organic acid ammonium salts, amines, peptone, meat extract, corn steep liquor, casein hydrolyzate, soybean meal Decomposed products, various fermented bacterial cells and digested products thereof are used.
[0015]
Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, magnesium chloride, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium chloride, and calcium carbonate.
The culture is performed under aerobic conditions such as shaking culture or aeration and agitation culture, and the culture temperature is 20 to 40 ° C, preferably 28 to 37 ° C. The pH of the medium is in the range of pH 5-9, preferably kept near neutral. The pH of the medium is adjusted with calcium carbonate, inorganic or organic acid, alkaline solution, ammonia, pH buffer solution, or the like. Usually, by culturing for 1 to 7 days, the amino acid according to the present invention is produced and accumulated in the culture solution.
[0016]
After completion of the culture, the amino acid can be recovered from the culture solution by removing precipitates such as cells from the culture solution and using an ion exchange treatment method, a concentration method, a salting-out method, or the like.
The amino acid obtained by the present invention is not particularly limited as long as it is an amino acid related to the present invention described above, and examples thereof include L-histidine.
Examples of the present invention are shown below, but the present invention is not limited to these examples.
[0017]
【Example】
Example 1 Acquisition of L-histidine-producing mutant having resistance to aminoquinoline derivative Based on the Budapest Treaty, dated March 9, 1999 as FERM BP-6673, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Japan) L-histidine-producing mutant H- with resistance to 1,2,4-triazolealanine derived from methionine-requiring Escherichia coli ATCC 21318, deposited in Tsukuba City, East 1-3-1 Tsukuba City, Ibaraki Prefecture 9340 was subjected to a mutation treatment (0.2 mg / ml, 30 ° C., 30 minutes) with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) according to a conventional method, followed by 150 mg of primaquinone disodium salt. / L-containing agar plate medium [glucose 0.2%, monopotassium phosphate 0.3%, disodium phosphate 0 .6%, Magnesium sulfate 0.01%, Sodium chloride 0.05%, Ammonium chloride 0.1%, Nutritional requirement substance (DL-methionine) 50 mg / L, Agar 1.5%, pH 7.2] .
[0018]
The agar plate medium coated with the cells was cultured at 30 ° C. for 2 to 6 days, and the large colonies that had grown were isolated by fishing to obtain H-9341. This strain was deposited under the Budapest Treaty on March 9, 1999 as FERM BP-6664 with the Institute of Biotechnology, Institute of Industrial Science and Technology (1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan). Yes.
Example 2 Growth Comparison Test on Agar Plate Medium Containing Primakin Growth of mutated strain H-9341 obtained in Example 1 on an agar plate medium containing primaquine was compared with the parent strain H-9340.
[0019]
To each mutant strain obtained when the aforementioned agar plate medium on containing primaquine disodium salt at the same concentration, the bacterial cells were cultured for 24 hours in a natural medium were suspended in physiological saline 1~10cells / cm 2 This was applied and cultured at 33 ° C. for 4 days.
Table 1 shows whether the culture can be grown.
The parent strain H-9340 could not grow on an agar medium containing primaquine.
[0020]
[Table 1]
[0021]
Example 3 Production of L-histidine L-histidine was produced by the following method using the mutant H-9341 obtained in Example 1 or the parent strain H-9340.
H-9340 and H-9341 were added to a seed medium (glucose 2%, molasses 0.5%, corn steep liquor 1%, ammonium sulfate 1.2%, monopotassium phosphate 0.3%, Magnesium sulfate 0.015%, DL-methionine 600 mg / L, adenine 100 mg / L, calcium carbonate 3%, pH 6.2) was inoculated into 6 ml, and cultured with shaking at 30 ° C. for 12 hours.
[0022]
0.1 ml of the seed culture solution thus obtained was added to the production medium (glucose 6%, corn steep liquor 1%, ammonium sulfate 2.4%, monopotassium phosphate 0.4%, magnesium sulfate 0. 015%, thiamine hydrochloride 10 mg / L, calcium pantothenate 10 mg / L, calcium carbonate 3%, pH 6.5) was inoculated into 5 ml, and cultured with shaking at 30 ° C. for 48 hours.
[0023]
After cultivation, the amount of L-histidine accumulated in the culture broth was quantified by high performance liquid chromatography.
The results are shown in Table 2.
The L-histidine productivity of the mutant H-9341 was improved as compared to the parent strain H-9340.
[0024]
[Table 2]
[0025]
【The invention's effect】
According to the present invention, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, glycine, L-serine, L-threonine, L-cysteine, L-tyrosine, Production ability of an amino acid selected from the group consisting of L-asparagine, L-glutamine, L-lysine, L-histidine, L-arginine, L-aspartic acid and L-glutamic acid (hereinafter abbreviated as amino acid according to the present invention) And obtaining a microorganism having resistance to an aminoquinoline derivative and culturing it in a medium, the productivity of the amino acid according to the present invention can be improved, and the amino acid according to the present invention is industrially efficient, And it can be manufactured at low cost.
Claims (6)
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP26510899A JP4245746B2 (en) | 1999-09-20 | 1999-09-20 | Amino acid production by fermentation |
CA002319282A CA2319282C (en) | 1999-09-20 | 2000-09-14 | Method for producing amino acids by fermentation |
US09/665,617 US7067289B1 (en) | 1999-09-20 | 2000-09-19 | Method for producing histidine by fermentation with E. coli |
AT00120126T ATE365212T1 (en) | 1999-09-20 | 2000-09-19 | METHOD FOR THE FERMENTATIVE PRODUCTION OF L-HISTIDINE USING AMINOQUINOLINE-RESISTANT BACTERIA STRAINS OF THE GENUS ESCHERICHIA |
EP00120126A EP1085087B1 (en) | 1999-09-20 | 2000-09-19 | Method for producing L-histidine by fermentation using aminoquinoline resistant bacterial strains of the genus Escherichia |
MXPA00009169A MXPA00009169A (en) | 1999-09-20 | 2000-09-19 | Method for preparing amino acid by fermentation. |
DE60035243T DE60035243T2 (en) | 1999-09-20 | 2000-09-19 | Process for the fermentative production of L-histidine by aminoquinoline-resistant bacterial strains of the genus Escherichia |
US11/414,175 US7871808B2 (en) | 1999-09-20 | 2006-05-01 | Isolated microorganism having an ability to produce L-histidine |
Applications Claiming Priority (1)
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JP26510899A JP4245746B2 (en) | 1999-09-20 | 1999-09-20 | Amino acid production by fermentation |
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JP2001086998A JP2001086998A (en) | 2001-04-03 |
JP4245746B2 true JP4245746B2 (en) | 2009-04-02 |
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JP26510899A Expired - Lifetime JP4245746B2 (en) | 1999-09-20 | 1999-09-20 | Amino acid production by fermentation |
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US (2) | US7067289B1 (en) |
EP (1) | EP1085087B1 (en) |
JP (1) | JP4245746B2 (en) |
AT (1) | ATE365212T1 (en) |
CA (1) | CA2319282C (en) |
DE (1) | DE60035243T2 (en) |
MX (1) | MXPA00009169A (en) |
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DE60035243D1 (en) | 2007-08-02 |
EP1085087A3 (en) | 2003-01-15 |
JP2001086998A (en) | 2001-04-03 |
US7067289B1 (en) | 2006-06-27 |
EP1085087A2 (en) | 2001-03-21 |
ATE365212T1 (en) | 2007-07-15 |
CA2319282C (en) | 2009-11-17 |
CA2319282A1 (en) | 2001-03-20 |
EP1085087B1 (en) | 2007-06-20 |
MXPA00009169A (en) | 2002-07-22 |
DE60035243T2 (en) | 2008-03-20 |
US7871808B2 (en) | 2011-01-18 |
US20060194302A1 (en) | 2006-08-31 |
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